1. Field of the Invention
The present invention relates to a novel DNA construction, wherein an insulator and a coat color reporter are introduced together into a mammalian genome to reduce the variability of a foreign gene expression in a transgenic mouse and to identify the genotype with a visible marker therein.
2. Description of the Related Art
For functional genetics analysis, the techniques of genetic modifications are often used in this field. But they're time consuming and laborious, especially in mouse system. Transgenic mouse producing by pronuclear microinjection is a system often used in genetic and clinical research, but low expression rate and labor genotyping are its disadvantages. Therefore, establishing a fast and useful system will be helpful the application of transgenic mouse in related research. PCR (polymerase chain reaction) and Southern analysis are most used in transgenic mouse genotype identification, but they're time consuming. A reporter gene within DNA construct can be a good marker to assist identification of transgenic mouse, but the variability of foreign gene expression caused by position effect is a major obstacle to hamper its development. In order to achieve the reporter system, a solution needs to be provided and applied to transgenic mouse model.
The following references provide more detailed description of the relevant art. The contents of these references are hereby incorporated by reference in their entirety.    Bell A C, West A G, Felsenfeld G. (1999) The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell. 98: 387–396.    Bell A C, West A G, Felsenfeld G. (2001) Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science. 291: 447–450.    Chung J H, Whiteley M, Felsenfeld G. (1993) A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74: 505–514.    Chung J H, Bell A C, Felsenfeld G. (1997) Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci U S A. 94: 575–580.    Dobie K, Mehtali M, McClenaghan M, Lathe R. (1997) Variegated gene expression in mice. Trends Genet. 13: 127–30.    Duhl, D. M., Vrieling, H., Miller, K. A., Wolff, G. L. and Barsh, G. S. (1994a) Neomorphic agouti mutations in obese yellow mice. Nat. Genet. 8: 59–65.    Duhl, D. M., Stevens, M. E., Vrieling, H., Saxon, P. J., Miller, M. W., Epstein, C. J. and Barsh, G. S. (1994b) Pleiotropic effects of the mouse lethal yellow (Ay) mutation explained by deletion of a maternally expressed gene and the simultaneous production of agouti fusion RNAs. Development, 120: 1695–1708.    Emery D W, Yannaki E, Tubb J, Stamatoyannopoulos G. (2000) A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci U S A. 97: 9150–9155.    Gerasimova T I, Corces V G. (2001) Chromatin insulators and boundaries: effects on transcription and nuclear organization. Annu Rev Genet. 35: 193–208.    Kucera G T, Bortner D M, Rosenberg M P. (1996) Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants. Dev Biol. 173: 162–173.    Pikaart M J, Recillas-Targa F, Felsenfeld G. (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev. 12: 2852–2862.    Prioleau M N, Nony P, Simpson M, Felsenfeld G. (1999) An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene. EMBO J. 18: 4035–4048.    Recillas-Targa F, Bell A C, Felsenfeld G. (1999) Positional enhancer-blocking activity of the chicken beta-globin insulator in transiently transfected cells. Proc Natl Acad Sci U S A. 96: 14354–14359.    Sambrook J and Russell D W. (2001). Molecular Cloning: A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York.    Taboit-Dameron F, Malassagne B, Viglietta C, Puissant C, Leroux-Coyau M, Chereau C, Attal J, Weill B, Houdebine L M. (1999) Association of the 5′HS4 sequence of the chicken beta-globin locus control region with human EF1 alpha gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits. Transgenic Res. 8: 223–235.    Tsai T F, Jiang Y H, Bressler J, Armstrong D, Beaudet A L. (1999) Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome. Hum Mol Genet 8:1357–1364.    Tsai T F, Chen K S, Weber J S, Justice M J, Beaudet A L. (2002) Evidence for translational regulation of the imprinted Snurf-Snrpn locus in mice. Hum Mol Genet. 11: 1659–1668.    Vassar R, Rosenberg M, Ross S, Tyner A, Fuchs E. (1989) Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc Natl Acad Sci U S A. 86:1563–1567.    Vassar R, Fuchs E. (1991) Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation. Genes Dev. 5: 714–727.    Wang Y, DeMayo F J, Tsai S Y, O'Malley B W. (1997) Ligand-inducible and liver-specific target gene expression in transgenic mice. Nat Biotechnol. 15: 239–43.    Wolff, G. L., Roberts, D. W., Morrissey, R. L., Greenman, D. L., Allen, R. R., Campbell, W. L., Bergman, H., Nesnow, S. and Frith, C. H. (1987) Tumorigenic responses to lindane in mice: potentiation by a dominant mutation. Carcinogenesis 8: 1889–1897